Purification process for 9alpha-fluorohydrocortisone compounds



PURIFICATION PROCESS FOR 9a-FLUOROHYDRO- CORTISONE COMPOUNDS Robert P.Graber, Elizabeth, and Calvin Stewart Snoddy, In, Plainfield, N. J.,assignors to Merck & Co., Inc., Rahway, N. J a corporation of New JerseyApplication December 7, 1956 Serial No. 626,811

6 Claims. c1. Mil-397.45)

No Drawing.

9a=fiuorohydrocortis0ue compound, are other steroids or steroid-likesubstances. The difliculty inrecovering all of the9a'-fluorohydrocortisone compound'formed is the primary cause of the lowyields which have heretofore been obtained in preparing pure9a-fiuorohydrocortisone compounds.

It is an object of the invention to provide a process for thepurification of 9a-fiuorohydrocortisone compounds.

It is another object to provide such a process whereby the undesirablecomponents of a crude reaction mixture, formed in the preparation of9a-fluorohydrocortisone compounds, are readily removed.

It is a further object of the invention to provide a purificationprocess for 9a-fluorohydrocortisone compounds which'will result ingreater yields of these compounds than were heretofore possible.

Other objects and the advantages of the invention will appearhereinafter.

In accordance with the invention, a crude 9a-fluorohydrocortisonecompound is purified by first reacting the crude mixture with anacylating agent and then subjecting the resulting acylated crude mixtureto a purification procedure such as crystallizing from an organicsolvent or chromatographic separation. By the term crude9afluorohydrocortisone compound is meant 9ot-fluorohydrocortisone or oneof its 2l-esters contaminated with other steroidal or steroid-likecompounds, which are present, "to some degree, in all reaction productsformed in the preparation of 9a-fluorohydrocortisone and its 21-esters.The 21-esters of 9a-fiuorohydrocortisone are those which have anacyloxygroup attached to the 21-carbon atom. The process of this invention isparticularly eifective with the 2l-esters wherein the ester has from oneto eight carbon atoms. Typical ester groups which maybe present areacetate, propionate, butyrate, benzoate, phenylacetate, t-butyl acetate,p-amino-benzoate, hydrocinnamate, succinate, a-naphthyl-acetate,a-naphthoate and caprylate.

I In the preparation of a 9u-fiuorohydrocortisone compound a crudemixture is obtained which contains, besides the 9a-fluorohydrocortisonecompound, other steroidal substances which may be small quantities ofthe starting material, as wellas other compounds whichlmay 2,876,238Patented Mar. 3, 1959 ice 2 be various fluorinated steroids ordegradation or rearrangement products. As for example, the 21-esters of9afiuorohydrocortisone may be prepared by reacting the corresponding21-ester of 9 9,ll 8-oxide-4-pregnene-l7a, 2l-diol-3,20-dione with asource of hydrogen fluoride. thereby forming the desired product inadmixture with other undesirable steroids, steroid-like substances, andpigments. When the- 2l-ester of 9B-l1l9-oxido-4-pregnen-17a,21-diol-3,20-dione is reacted as above indicated with hydrogenfluoride, in addition to the desired 21-ester of9ot-fluorohydrocortisone, there are obtained significant quantities ofthe corresponding A804) compound, Apregnadiene-l1,l7u,2l-triol-3,20-dione, which has been identified as thepredominating by-product of this reaction.

The reaction product containing the desired 9a-fluorohydrocortisonecompound and the undesired N compound is acylated with a suitableacylating agent as defined hereinbelow. The desired product, that is,the 9afluorohydrocortisone compound, is unaifected by the acylatingagent, whereas the undesired A compound is converted to' its ll-acyloxyderivative. While the fluorohydrocortisone compound and the A compoundhave physical properties such that separation of these compounds fromeach other is extremely difiicult, separation of the9a-fiuorohydrocortisone compound from the ll-acyloxy derivative of theA804) compound is relatively simple to accomplish by conventionaltechniques such as crystallization, chromatography, and the like.

The acylation of the crude mixture is carried out by admixing with anacylating agent. Suitable acylating agents are organic acid halides,organic acid anhydrides, ketenes, organic esters, and others, with theacid anhydrides being preferred, the acylating agent containing theacylresidue of an organic earboxylic acid, especially those containingfrom one to eight carbon atoms, inclusive. Among suehacids are formic,acetic, propionic, butyric, valeric, hexanoic, heptanoic, octanoic,cyclopentane-carboxylic, cyclopentylpropionic, benzoic, toluic, oxalic,and the like. Usually an excess of the acylating agent is used. However,any quantity from about the theoretical amount to about times thisamount, or even more, is operative, with an excess of about two to aboutten times the theoretical amount being preferred. The acylation may beelfected in a solvent, such as ethers, chlorinated hydrocarbons orhydrocarbons or the acylating agent may act as the solvent. Typicalexamples of such solvents are chloroform, benzeneand toluene. A smallamount of a tertiary amine catalyst such as pyridine, collidine andtrimethylamine may be added when an organic acid anhydride or a keteneis the acylating agent and the amine catalyst may also act as thesolvent. When organic esters are used it is desirable to use an acidcatalyst such as sulfuric acid or p-toluenesulfonic acid. The mixture isheated, usually at about 25 to 100 (3., lower or higher temperaturesalso usually being operative. A reaction period of about one to twentyhours is satisfactory, with a period of about ten to sixteen hoursbeingpreferred at room temperature and from five to sixty minutes at 50 C.

The 9a-fluorohydrocortisone compound may be purified in any convenientmanner. For example, if most of the acylating agent has been consumed.during the reaction, the last traces may be removed under vacuum, theresidue dissolved in an organic solvent such as ether, chloroform, andthe like, and the resulting solutionwashed with an aqueous base such asdilute aqueous sodium bicarbonate, sodium carbonate, sodium hydroxideand the like with a dilute aqueous acid to remove any amine catalyst.The

washedjsolution istreated with a drying-agent such as sodium sulfate,the solution thus treatedfiltered to removethe 7 either by gravity orunder slight pressure.

, 3 drying agent, the solvent distilled, and the residue crystallizedfrom a suitable solvent such as acetone. Alternately, if the acylatingagent has not been consumed, the reaction mixture may be stirred withwater or an aqueous base such as aqueous sodium bicarbonate solution-todecompose the excess acylating agent. The resulting mixture is extractedwith an organic solvent, the organic extract washed with an aqueousbase, with a dilute aqueous acid and then with water until neutral andthe product isolated from the washed organic solution.

The 9a-fluorohydrocortisone compound may be crystallized from any of thefollowing solvents: benzene, acetone-petroleum ether, acetone-ether,chloroform-benzene, ethyl acetate-ether and ethyl acetate-petroleumether.

The acylated crude mixture containing the 9ot-fiuorohydrocortisonecompound can be purified or the crystal line material obtained by theabove procedure can be further purified by adsorbing on an essentiallyneutral adsorbent material preferably in a column of the chromatographictype, washing the adsorbent material with solvent to develop in thecolumn zones of adsorbent material containing the various components ofthe crude mixture in differing amounts, and differing degrees of purity,and selectively recovering the 9a-fluorohydrocortisone compound fromsaid zones of adsorbent material.

7 Various essentially neutral adsorbents can be utilized in thepracticeof this invention, including neutral alumina, silica gel, silicic acid,activated carbon, Fluorosil (an adsorbent prepared according to U. S.Patent No. 2,393,625), and the like; neutral alumina having a pH of fromto 8 and more preferably 6.5 to 7.5. The adsorbent is preferablycontained in a column of the chromatographic type. If desired, the rateof flow of eluting solvent can be modified by adding a material such asfilter paper pulp to the adsorbent or by pressure.

In preparing the adsorbent, such as alumina, it is treated so that whenWashed with water the washes will have a pH of about 5 to about 7. Acolumn is packed with an amount of adsorbent within the range of aboutto 200 grams, preferably to grams, for each gram of the crude mixture orisolated product to be treated. The column is preferably filled withsolvent and dry adsorbent sifted into the solvent until the column ispacked.

The crude mixture containing the 9a-fluorohydrocortisone compound isthen dissolved in a non-polar solvent for the mixture to form aconcentration of about 2 to 50%. The solution should be as concentratedas possible and the solvent as non-polar as possible. The solution ispoured onto the upper surface of the adsorbent in the column, andallowed to fiow into the adsorbent either by gravity or under pressure.When the solution of the crude material vis all in the adsorbent, freshsolvents not necessarily the same as that originally used, and generallysolvents with increasingly higher polarity, are added to the top of thecolumn and allowed to percolate through After solids appear in theeluate, various fractions of eluate are collected at arbitrary orpredetermined intervals. An empiracal guide as to the fractions ofeluate to be collected can be readily worked out for any particularcrude material to be treated by making a few preliminary runs,collecting fractions of eluate at arbitrary intervals and determiningthe amounts of the desired product in the various fractions. The9a-fluorohydrocortisone compound usually remains on the adsorbent untilall the major impurities, and most of the pigments, are eluted. The9a-fluorohydrocortisone compound is isolated from the collectedfractions of eluate by concentrating to dryness and recrystallizing froma solvent such as those mentioned above.

Typical solvents for the crude 'acylate mixture or crystalline materialcontaining the 9oc-flll0l0hYdf0C0l'tisone compound which may bementioned, are chloroform, chloroform-benzene, acetone-petroleum ether,acetone ether, chloroform-petroleum ether, ethylene dichloride,

carbon tetrachloride and mixtures of these solvents with benzene.

The solvent media used for developing the zones or hands of differentcompositions in the column of adsorbent include generally solvents ofincreasing polarity. Examples of suitable solvents are benzene,benzene-chloroform, chloroform, chloroform-ether, ether and the like. Itis sometimes advantageous in the adsorption step to employ a solventmedium in which the 9rz-fiuorohydrocortisone compound is not too readilysoluble in order that better distribution of adsorbate in the column canbe obtained before any appreciable amount of the 9a-fiuorohydrocortisonepasses from the column. For these purposes benzene, chloroform, ethylenedichloride mixtures of chloroform and benzene or chloroform and ethylenedichloride can be employed.

An alternate procedure for obtaining separate fractions from the columnis to drain the column of solvents, divide the adsorbent into separatesections, known or determined to contain optimum or predominant amountsof 9a-fluorohydrocortisone compound, and then to extract the separatedsections with a suitable solvent and isolate the9cc-fil10lOhYdl'OCOlllS0l16 in the manner previously described.

The distribution of bands or sections of adsorbed 9afiuorohydrocortisonecompound in the column may differ depending upon the composition of thecrude mixture containing the 9a-fluorohydrocortisone being purified. Thefirst fraction of eluate or lower zones, bands or sections of the columnusually contain the major impurities, and some of the pigments and laterfractions of eluate or higher bands or sections of the column containpredominant amounts of the 9a-fiuorohydrocortisone compound.

It is desirable in all the methods to identify the desired fractions byrunning paper chromatographs of the various fractions.

It will be understood that the degree of purity or potency of the9a-fiuorohydrocortisone will depend in large measure upon the care takenin determining the amounts of the different fractions of eluate to becollected or similarly the particular manner in which a column ofadsorbent with adsorbed 9u-fluorohydrocortisone compound is divided forsubsequent extraction of separate parts thereof. It will further beunderstood that by subjecting the 9a-fiuorohydrocortisone compound,

. recovered from the various eluate fractions to additional selectiveadsorption and elution treatment, products of higher purity can beobtained.

The following examples are given for purposes of illustration:

Example 1 To an ice-cold solution of 0.572 gram (28.60 millimoles) ofanhydrous hydrogen fluoride in 220 ml. of ice-cold alcohol-freechloroform was added an ice-cold solution of 2.88 grams (7.15millimoles) of 95,11,6- oxido- 4 pregnene 170:,21 diol 3,29 dione -21-acetate in 40 ml. of alcohol-free chloroform. The mixture was allowed tostand at 0 C. for four and onehalf hours. Then ml. of water was added,the mixture shaken and the layers separated. Washing with water wascontinued until neutral, and then the chloroform layer was dried and thechloroform removed in vacuo to afford 3.32 grams of amorphous residue.Alternatively, the final reaction mixture is treated with aqueous sodiumacetate solution to neutralize the excess hydrogen fluoride, then washedwith water until neutral, and then the chloroform layer dried and thechloroform removed in vacuo to afford the amorphous residue.

Example 2 The 3.32 gram-sample, prepared in Example 1, was acetylatedwith 10 ml. of pyridine and 10 ml. of acetic anhydride overnight at roomtemperature. After decomposing the excess acetic anhydride with ice andwater, the organic material was extracted with several portions of ethylacetate. The combined ethyl acetate extracts were washed with water, anddilute hydrochloric acid to remove the pyridine, again with water, thenwith dilute sodium bicarbonate until the washes were alkaline, finally.with water and saturated salt solution, dried, and the solvent removedin vacuo to afford an amorphous residue, weight=3.34 grams. The materialwas crystallized from chloroform-benzene to afford the product.

Example 3 The 3.34 gram-sample, prepared in Example 2, waschromatographed over 165 grams of an adsorbent comprising about 84% SiO15% MgO and 1% Na SO The fractions obtained by elution with chloroformand 5% acetone in chloroform were concentrated to dryness and theresidues which crystallized on addition of benzene were combined andevaporated to dryness in vacuo, weight=1.278 grams. Recrystallizationfrom chloroform-benzene afforded 1.189 grams of substantially pure9a-fluorohydrocortisone acetate as the one-half mole benzene solvate,melting point 225-227 0., soft at 223 C.

Example 4 The crude acetylated product, prepared as in Example 2, waschromatographed over neutral alumina (ca. 40-50 gram/gram acetylatedproduct). The fractions eluted with 50% chloroform-benzene and withchloroform were evaporated to dryness in vacuo. Each residue wasanalyzed by paper chromatography. The residues containing the9a-fluorohydrocortisone compound on recrystallization fromchloroform-benzene and from acetonepetroleum ether (boiling point 60-70C.) afiorded the substantially pure product.

Any departure from the above description, which conforms to the presentinvention, is intended to be included within the scope of the claims.

What is claimed is:

1. A process for purifying a crude mixture containing a9a-fluorohydrocortisone compound selected from the group consisting of9a-fluorohydrocortisone and 21- acyloxy derivatives thereof, wherein theacyl group is derived from an organic carboxylic acid containing fromone to eight carbon atoms, and a A -pregnadiene-11,17oc,21-t1'i01-3,20-di0116 compound selected from the groupconsisting of A -pregnadiene-11,17u,21-triol-3, 20-dione and 21-acyloxyderivatives thereof, wherein the acyl group is derived from an organiccarboxylic acid containing from one to eight carbon atoms, whichcomprises: treating the crude mixture with an acylating agent containingthe acyl residue of an organic carboxylic acid containing from one toeight carbon atoms, separating the unused acylating agent from theresulting mixture containing unreacted 9a-fluorohydrocortisone compoundand the ll-acyloxy derivative of the A -pregnadiene-11,17u,21-triol-3,20-dione compound, and crystallizing the9a-fiuorohydrocortisone compound from the resulting mixture containingthe acylated A -pregnadiene- 11,17a,21-triol-3,20-dione compound.

2. The process of claim 1 wherein the 9oa-fill0l'0hYdI0- cortisonecompound is 9a-fiuoro-4-pregnene-l1,B-17a,21-triol-3,20-dione-2l-acetate.

3. The process of claim 1 wherein the acylating agent is a mixture ofacetic anhydride and pyridine.

4. A process for purifying a crude mixture containing a9a-fluorohydrocortisone compound selected from the group consisting of9a-fiuorohydrocortisone and 21- acyloxy derivatives thereof, wherein theacyl group is derived from an organic carboxylic acid containing fromone to eight carbon atoms, and a A -pregnadiene-11,17a,21-triol-3,20-dione compound selected from the group consistingof A -pregnadiene-11,17a,21-triol- 3,20-dione and 21-acyloxy derivativesthereof, wherein the acyl group is derived from an organic carboxylicacid containing from one to eight carbon atoms, which comprises:treating the crude mixture with an acylating agent containing the acylresidue of an organic carboxylic acid containing from one to eightcarbon atoms, separating the unused acylating agent from the resultingmixture containing unreacted 9a-fluorohydrocortisone compound and thell-acyloxy derivative of the A -pregnadiene-l1,17a,21-triol-3,20-dionecompound, and separating the 9u-fluorohydrocortisone compound from theresulting mixture containing the acylated A-pregnadiene-l1,17a,21-triol-3,20-dione compound by chromatographicmeans.

5. The process of claim 4 wherein the 9a-fluorohydrocortisone compoundis 9a-fluoro-4-pregnene-11B-17a,21- triol-3,20-dione-2l-acetate.

6. The process of claim 4 wherein the acylating agent is a mixture ofacetic anhydride and pyridine.

Journal of the American Chemical Society," 76, pages 1455-1456 March1954, Fried et a1.

1. A PROCESS FOR PURIFYING A CRUDE MIXTURE CONTAINING A9A-FLUOROHYDROCORTISONE COMPOUND SELECTED FROM THE GROUP CONSISTING OF9A-FLUOROHYDROCORTISONE AND 21ACYLOXY DERIVATIVES THEREOF, WHEREIN THEACYL GROUP IS DERIVED FROM AN ORGANIC CARBOXYLIC ACID CONTAINING FROMONE TO EIGHT CARBON ATOMS, AND A $4.3(14)-PREGNADIENE11,17A,21-TRIOL-3,20-DIONE COMPOUND SELECTED FROM THE GROUP CONSISTING OF$4,3(14)-PREGNADIENE-11,17A,21-TRIOL-3, 20-DIONE AND 21-ACYLOXYDERIVATIVES THEREOF, WHEREIN THE ACYL GROUP IS DERIVED FROM AN ORGANICCARBOXYLIC ACID CONTAINING FROM ONE TO EIGHT CARBON ATOMS, WHICHCOMPRISES: TREATING THE CRUDE MIXTURE WITH AN ACYLATING AGENT CONTAININGTHE ACYL RESIDUE OF AN ORGANIC CARBOXYLIC ACID CONTAINING FROM ONE TOEIGHT CARBON ATOMS, SEPARATING THE UNUSED ACYLATING AGENT FROM THERESULTING MIXTURE CONTAINING UNREACTED 9A-FLUOROHYDROCORTISONE COMPOUNDAND THE 11-ACYLOXY DERIVATIVE OF THE$4,3(14)-PREGNADIENE11,17A,21-TRIOL-3,20-DIONE COMPOUND, ANDCRYSTALLIZING THE 9A-FLUOROHYDROCORTISONE COMPOUND FROM THE RESULTINGMIXTURE CONTAINING THE ACYLATED$4,3(14)-PREGNADIENE11,7A,21-TRIOL-3,20-DIONE COMPOUND.